Assessment of the cryosurvival of Spanish horse spermatozoa cryopreserved by microdroplets and straw vitrification techniques

Vitrification (VIT) of equine spermatozoa could be a rapid and cost-effective alternative to conventional slow freezing (CS). Vitrification prevents ice crystal formation and preserves cellular integrity. Cryosurvival outcomes following VIT differ between small-volume 30-µL microdroplets (VIT-MD) and larger-volume 250-µL straws (VIT-ST). In this context, this study compared sperm cryosurvival after CS freezing and 2 VIT protocols (VIT-MD and VIT-ST). For this purpose, 16 ejaculates were collected from four healthy Spanish stallions (four per horse) between February and March in a high-tropical Ecuadorian breeding facility (3°14′35.5″S, 79°14′41.8″W). After initial dilution in Botusemen-Gold® extender, each ejaculate was split into 3 aliquots for conforming to 3 treatments, respectively: CS (as control), VIT-MD, and VIT-ST. The CS protocol included 5% dimethylformamide (DMF), loading into 0.5-mL straws, and exposure to liquid nitrogen (LN2) vapor (Tamay et al. 2023 Biopreservation and Biobanking 21, 561–568). Both VIT protocols used 100 mM trehalose as a non-permeable cryoprotectant agent. In VIT-MD, 30-µL microdroplets were directly plunged into LN2, forming spherical beads. In VIT-ST, 250-µL samples were loaded into straws, inserted into 500-µL outer straws, and vitrified by horizontal immersion in LN2. All samples were adjusted to 50 × 106 sperm/mL. Thawing and warming conditions were as follows: CS (n = 48 straws), 37°C water for 30 s; VIT-MD (n = 48 cryovials), droplets over a 65°C plate for 5 s; and VIT-ST (n = 48 straws), straws in 3 mL of Botusemen-Special at 43°C for 20 s. Vitrified samples were centrifuged (300 × g for 5 min) and resuspended in Botusemen-Gold® extender at 37°C before sperm analysis. A one-way ANOVA followed by Bonferroni post hoc tests assessed sperm kinematics, plasma membrane integrity, and head morphometry, with stallion as covariate. The results showed that all cryopreservation methods significantly impaired motility versus fresh semen (P < 0.05). Nonetheless, the CS group maintained significantly higher total (TM) and progressive (PSM) motilities, curvilinear (VCL) and straight-line (VSL) velocities, straightness (STR), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), viability, and acrosomal integrity compared with VIT-MD and VIT-ST (P < 0.05). Among VIT methods, VIT-MD achieved superior kinematic (e.g. motilities, velocities, ALH, and BCF), viability, and acrosome integrity preservation relative to VIT-ST (P < 0.05). Morphometric analysis showed that head area and circumference increased after VIT-MD or VIT-ST compared with fresh and CS frozen samples (P < 0.05). We propose that the higher cryoresponse of the VIT-MD method was attributable to the smaller volume of the samples together with the warming procedure (65°C/5 s); it is suggested that under these conditions, the risk of recrystallization during warming was reduced. In conclusion, although conventional slow freezing was more effective than vitrification, microdroplet vitrification produced greater sperm cryosurvival and quality than straw vitrification for preserving Spanish horse sperm.